**3.2 Interaction of tetraspanins with integrins and matrix-degrading enzymes: role of tetraspanins in cancer and metastasis**

Important association partners of tetraspanins are the integrins. The role of such complexes in invasive growth *in vivo* as well as the effect of integrin-mediated binding events on cell proliferation and invasion is well established. Especially, the laminin-binding integrins (α6β4, α3β1, α6β1 and α7β1) exhibit extensive interactions with tetraspanin proteins [12, 38]. The functionality of integrins may depend critically on their interaction of tetraspanins: it has early been described that the remarkably stable association of the tetraspanin CD151 and the integrin α3β1 leads to a high level of activation of cellular PI4K [39]. Further, CD151 interacts directly with the α<sup>3</sup> subunit and links it to other tetraspanins, CD9 and CD81. Loss of CD151 abrogates the α3β1 mediated mobility on its ligands, laminin-332 and laminin-551. CD9/CD81 complex may even regulate the integrin-mediated functions independently of CD151 by forming a complex with the integrin and directing the PKCα-α3β1 association [40]. Another example of tetraspanin-integrin association reveals its proangiogenic role through VEGF induction, mediated by cooperation between TM4SF5 and integrin α5 of epithelial cells [41]. Interestingly, removal of CD151 palmitoylation sites did not disrupt the CD151–α6β4 complex in epithelial cells but strongly influenced α6β4 integrin–dependent cell morphology [42]. The rat tetraspanin D6.1A (human homolog is CO-029) was able to induce systemic angiogenesis by initiation of an angiogenic loop that reached organs distant from the tumor, probably due to the abundance of D6.1A in tumor-derived exosomes. This is in line with reports claiming that EVs prepare niches for metastatic tumor cells at tissues distant from the primary tumor [43]. This tetraspanin associates with integrins α3β1, α6β1 and α6β4, as well as with tetraspanins CD9 and CD81, and is similarly to CD151 linked to tumor-promoting activities [44].

Active complexes of tetraspanins and integrins influence biological processes other than cellular signaling by interacting with cellular metalloproteinases, important players in the remodeling of extracellular matrix. A study of MDA-MB-231 cells, a breast cancer cell line, has indicated that the α3β1-tetraspanin protein complex may be linked to an invasive phenotype of tumor cells via modulation of various signaling pathways, including activation of membrane metalloproteinase-2 (MMP-2), an enzyme associated with invasive migration of the cells, and affecting phosphatidylinositol-3-kinase (PI3K) signaling pathways, which control actin cytoskeleton dynamics [45]. By the incorporation of the members of a disintegrin and metalloproteinase (ADAM) family members the tetraspanins are able to influence the

*Current Topics in Biochemical Engineering*

association partners as shown for CD9 [33].

**3. Natural ligands of tetraspanin proteins**

**3.1 The tetraspanin web and intertetraspanin contacts**

tions, while speculating how this might translate to EVs.

and C-terminal transmembrane cysteines to serine eliminated palmitoylation of CD151, which turned out to be deleterious for the assembly with other cell surface proteins, including tetraspanins CD9 and CD63, their organization to TEMs and subsequently their subcellular distribution and cell morphology [28]. At the same time, it had minimal influence on the density of tetraspanin protein complexes and was dispensable for CD151-α3β1 integrin association. Depalmitoylation of CD81 did not impact its surface expression and stability, but rendered it less available for contact with its natural interaction partner CD9 and the relevant epitopes less

The four transmembrane helices of tetraspanin proteins form two largely separated pairs of antiparallel helices: one pair comprises TM1/TM2 and the other TM3/TM4. The two pairs of helices only converge close to the cytoplasmic side of the membrane through contacts between TM2 and TM3. In the recently solved crystal structure of full-length CD81, this cone-like structure has been shown to harbor a binding pocket for cholesterol [30], and mutations within transmembrane domains in certain tetraspanins have been connected with pathological states [31]. The short cytoplasmic tails show no obvious functional significance in signaling processes, suggesting that their signaling competence relies on association with other molecules [32]; nevertheless, its mutation can lead to different assembly with

The ability of members of the tetraspanin family to assemble into a unique biological feature known as tetraspanin-enriched microdomain (TEM) is due to their mutual interactions; however, these structures include also receptors, integrins and signaling molecules such as phosphatidyl-kinase C (PKC) and phosphatidylinositol-4-kinase (PI4K) [9]. These interactions are fundamental for cellular functions such as cell adhesion, proliferation and motility. Interactions between tetraspanin members are important in maintaining the integrity and stability of the tetraspanin web and providing binding sites for different ligands. The multimers of newly synthesized proteins are formed in the Golgi apparatus. The predominantly cross-linked tetraspanin species are homodimers, but also higher order complexes and low amounts of heterodimeric tetraspanins (CD81/ CD9, CD9/CD151, CD81/CD151) were identified [27]. It has been suggested that tetraspanin homodimers, formed in the Golgi and present at the cell surface, serve as building blocks in the assembly of higher organized tetraspanin protein complexes. Interestingly, the exosomes originating from cell lines overexpressing CD9 are believed to be enriched in more stable TEMs [10]. Overall, although most tetraspanins can interact with most other tetraspanins, and similarly engage with several other proteins, the nature of these interactions has been until recently classified only according to their stability in the presence of detergents of different stringency, which does not necessarily reflect their significance in the cellular milieu [34]. A thorough characterization of strength and abundance of the interactions between the members participating in a tetraspanin web in a particular cell and physiological situation is therefore needed and will support the understanding of its mediated biological effects. Similarly, most data on tetraspanin functionality come from studies on their localization on cell membranes, while functional data in vesicles are still scarce. Therefore, we here summarize the known cellular func-

accessible for binding of structurally dependent antibodies [29].

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cellular ectodomain cleavage and release activity of these enzymes [46]. The tetraspanins of the TspanC8 group (tetraspanins with 8 cysteines) have a significant impact on the cellular exit and catalytic activity of ADAM10 [47], in particular the activity of Tspan15/ADAM10 promoted N-cadherin cleavage [48–50]. Different TspanC8/ ADAM10 complexes seem to have different substrate specificities [51]. The silencing of CD9 enhanced shedding of ADAM17-substrates TNF-α and ICAM-1 [52].

Important discovery of the possible consequences of fine differences in composition of TEMs has been delivered by the study of exosomes enriched in Tspan8-α<sup>4</sup> complex that were preferentially taken up by the endothelial and pancreatic cells [53]. The fact that such modifications can allow selective targeting *in vitro* and *in vivo* holds promise to achieve improved exosomal delivery by engineering of their membrane components.

#### **3.3 The role of tetraspanins in immune complexes**

In antigen-presenting cells (APCs), tetraspanins integrate into TEMs proteinrecognition receptors binding to conserved repeated motifs of microbes, such as Toll-like receptors, and MHCII molecules into tetraspanin web platforms, as well as Fcγ receptor I in phagocytic cells, Fcγ receptor IIb and III upon the activation of macrophages and Fcε receptor I in monocytes and skin-derived dendritic cells [54].

The particular role of CD81 protein in the formation of specialized microdomains in the plasma membrane of the cells of the immune system was discovered by elucidating its function of recruiting various adhesion molecules, receptors and signaling proteins to the central zone of the immune synapse in T-lymphocytes and APCs [55]. Therefore, it has early been proposed for CD81 to play a key role during antigenic presentation, since it colocalizes with the T-cell receptor/CD3 [56], and CD81 indeed turned out to be a regulator of CD3 clustering and sustained CD3 signaling [57].

Further, the T-cell side of the immune synapse is densely populated by tetraspanins CD9 and CD151. The abolishment of their expression reduces markers of activation of T-lymphocytes conjugated to the APCs, such as IL-2 secretion and expression of CD69 [58].

Another role in the immune response of tetraspanin CD81 is amplifying and sustaining B-cell receptor signaling from lipid rafts by ligation to the co-receptor CD19/CD21 complex. The signaling through a variety of cell surface protein complexes implies a role of lipid rafts, again highlighting the ability of tetraspanin to facilitate raft association [9].

#### **3.4 Tetraspanins in pathogen infection**

Several studies have been oriented towards the research of tetraspanins as ligand molecules for pathogen entry. CD81 has been identified as a ligand for hepatitis C virus (HCV) recognition and viral entry [59]. The ligand for viral glycoprotein E2 is the D-domain of the LEL, a dynamic region positioned within the triple-bundle helix, whose conformation in solution differs substantially from the one suggested by crystal structure. Challenging for structure-based design, this region nevertheless presents an attractive target for design of therapeutically relevant ligands with methods such as NMR [60]. Apart of the EC2 domain, other regions of CD81 have proven important for virus infection. Experimental evidence here was based on the exchange of the structural domains of the molecule with the ones of tetraspanins of different degrees of homology, and it was found that closely related substitutions were more efficient at functional complementation of CD81. Viral entry has been shown to correlate with surface expression of the chimeric protein and to depend on the presence of the cholesterol-coordinating glutamate residue [61]. EWI-2wint,

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*Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting*

a cleavage form of EWI-2, a member of the immunoglobulin superfamily, has an inhibitory effect on HCV infection by obstructing the interaction between CD81 and HCV E2 [62]. The related factor EWI-F inhibits *Plasmodium* infection, whereas

Tetraspanin microdomains have been described to regulate HIV-1 entry, assembly and transfer between the cells [64, 65]. CD81 influences importantly the early stages of virus replication by controlling the stability of HIV-1 restriction factor and consequently the activity of viral reverse transcriptase [66], while CD63 facilitates endocytosis of the HIV receptor CXCR4 [67] as well as supports the replication steps in macrophages [68, 69]. Also Coronaviruses and low-pathogenicity Influenza A viruses utilize TEM domains as entry portals to co-engage with cellular receptors and proteases, which enable viral proteolytic priming [70]. As shown in an *in vivo* model with CD151 null-mice, this tetraspanin is a critical novel host factor of nuclear export signaling of Influenza A virus, used complementary to the viral nuclear export proteins [71]. Tspan9 modulates the early endosome compartment to make it more permissive for membrane fusion of early-penetrating viruses, and its depletion strongly inhibits infection by alphaviruses that fuse in early endosomes but does not alter the delivery of virus to early endosomes or change their pH or protease activity [72]. It is unclear, what function then EV-based tetraspanins might have in the context of viral infection, and it might be speculated that cells use EVs to titrate away virus into membrane structures that are unable to provide replication and protein synthesis machineries for the virus. This is supported by the findings that EVs might

*DOI: http://dx.doi.org/10.5772/intechopen.83537*

its silencing increases infection efficiency [63].

have anti-influenza infection activity *in vitro* [73].

**interactions**

**4. Extracellular vesicles as mediators of cell-cell interaction**

**4.1 Biological basis for therapeutic applications: EVs as mediators of intercellular** 

EVs are secreted by most cell types and are taken up by recipient cells, where their cargo consisting of a cocktail of proteins, mRNAs and non-coding RNAs alters the behavior of the recipient cells in a way that might be even considered similar to hormones or cytokines [74], e.g. in the context of skin or bone cell paracrine signaling [75–77]. siRNAs (small interfering RNAs) and miRNA-based inhibitors have been recognized as potent novel drug candidates for many years. As EVs can be loaded with different drugs *in vitro*, they qualify as an attractive drug delivery system. The specificity of the recipient cell targeting *in vivo* is understood in a limited way only, although there is evidence of accumulation of specific EVs [43]. For example, EVs from human mesenchymal stem cells accumulated in the liver, spleen and sites of acute kidney injury [78]. Such tropism for a specific cell type, a requirement for targeted drug delivery, appears to be determined by surface proteins of the source cells. The composition of EV membrane reflecting the one of their source cell makes these particles non-immunogenic, and their small size allows them to pass the immune surveillance of the host organism [79, 80]. The reported engagement of exosomes in physiological processes in normal and diseased central nervous system makes them attractive vehicles for delivering neurotherapeutic agents across the blood-brain barrier [81–83]. Nevertheless, their delivery in humans seems so far limited to liver and kidney as they are reported not to reach therapeutic amounts in brain, heart and other tissues due to lack of specific targeting and thus low enrichment of the intended therapeutic ingredient in the target tissue. Modifications of the EV surface membrane to achieve enhanced targeting of a specific cell type are hence a common strategy embodied in several different engineering approaches.

*Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting DOI: http://dx.doi.org/10.5772/intechopen.83537*

a cleavage form of EWI-2, a member of the immunoglobulin superfamily, has an inhibitory effect on HCV infection by obstructing the interaction between CD81 and HCV E2 [62]. The related factor EWI-F inhibits *Plasmodium* infection, whereas its silencing increases infection efficiency [63].

Tetraspanin microdomains have been described to regulate HIV-1 entry, assembly and transfer between the cells [64, 65]. CD81 influences importantly the early stages of virus replication by controlling the stability of HIV-1 restriction factor and consequently the activity of viral reverse transcriptase [66], while CD63 facilitates endocytosis of the HIV receptor CXCR4 [67] as well as supports the replication steps in macrophages [68, 69]. Also Coronaviruses and low-pathogenicity Influenza A viruses utilize TEM domains as entry portals to co-engage with cellular receptors and proteases, which enable viral proteolytic priming [70]. As shown in an *in vivo* model with CD151 null-mice, this tetraspanin is a critical novel host factor of nuclear export signaling of Influenza A virus, used complementary to the viral nuclear export proteins [71].

Tspan9 modulates the early endosome compartment to make it more permissive for membrane fusion of early-penetrating viruses, and its depletion strongly inhibits infection by alphaviruses that fuse in early endosomes but does not alter the delivery of virus to early endosomes or change their pH or protease activity [72]. It is unclear, what function then EV-based tetraspanins might have in the context of viral infection, and it might be speculated that cells use EVs to titrate away virus into membrane structures that are unable to provide replication and protein synthesis machineries for the virus. This is supported by the findings that EVs might have anti-influenza infection activity *in vitro* [73].
